1. Field of the Invention
The invention relates to chemical compositions and their use in the culturing of microorganisms and tissues and more particularly relates to compositions and methods useful for culturing oxygen sensitive (anaerobic and . microaerophilic) microorganisms, and microorganisms and tissues requiring special gaseous environments.
2. Brief Description of the Prior Art
The prior art literature is replete with descriptions of methods and apparatus useful for culturing oxygen sensitive microorganisms. Representative of such descriptions are those found in the U.S. Pat. Nos. 2,348,448; 2,361,992; 2,463,143; 3,165,450; 3,208,909; 3,248,302; 3,483,089; 3,913,564; 4,030,978; 4,033,826; 4,038,148; and 4,200,610.
In spite of the many prior art methods and the large number of different apparatus previously described for culturing oxygen sensitive microorganisms, none have been completely satisfactory for all circumstances of needs and uses For example, a large number of methods and apparatus depend on the generation and use of hydrogen gas to combine with and thereby remove oxygen from the atmosphere; see for example U.S. Pat. No. 3,483,089. Of course, hydrogen gas poses the hazard associated with any explosive gas. In addition, relatively expensive catalysts may be required to satisfactorily promote the desired reduction of oxygen.
The apparatus and method of the present invention are not dependent on the generation or use of explosive gases or exotic catalysts and therefore obviate these problems of the prior art.
Many of the prior art methods of maintaining anaerobiosis within the confines of a culture environment depend on the use of special laboratory hardware such as anaerobic chambers, anaerobe jars and the like. In U.S. Pat. No. 2,348,448 there is described a modified Petri type of culture dish for use in culturing oxygen sensitive microorganisms. The cover of the dish is modified by depression of the central portion towards the bottom of the dish. This greatly reduces the air space within the closed dish and above any nutrient media disposed in the dish bottom. A. deeper annular depression near the peripheral rim of the cover contacts the upper surface of the nutrient media to seal the edges of the air space. The air space, sealed off, is about 1 mm deep. Oxygen in the air space is removed chemically by the presence of a reducing agent incorporated in the nutrient media (anaerobic agar).
Those individuals responsible for budgeting laboratory finances appreciate that the above-described special or modified forms of apparatus are relatively expensive. In the method of the present invention, using the compositions and apparatus of the invention, small laboratories with limited funds need not invest in the more expensive prior art equipment in order to carry out anaerobic microbiology culturing. Instead, they may use the ordinary, commercially available Petri dish with the compositions of the invention. No modification of the standard Petri dish is required. No special seals or "O" rings need be purchased to seal the culturing environment. In fact, the microbiologist need not modify either the equipment normally on hand in the laboratory or the technique of culturing in order to practice the present invention. This latter point is of particular importance since it means that laboratory personnel need not undergo specific training or retraining to practice the method of the invention.
The U.S. Pat. No. 3,248,302 includes in its description of the art a discussion of the so-called "single plate method" of growing strictly anaerobic bacteria. The term "single plate method" refers in particular to the use of a sealed Petri dish containing a nutrient medium, the surface of which is inoculated with the anaerobe desired for culture. After discussing in detail the problems associated with all the prior art methods of culturing anaerobes, including the single plate method, the patentee states that "the criteria for an ideal single plate method of growing bacteria under anaerobic conditions are generally as follows: that the method can be used routinely, that anaerobiosis (removal of oxygen) or other desired atmospheric conditions will result rapidly upon closing the culture dish or plate; that in the practice of the method, any culture medium suitable to culturing the bacteria in a standard size Petri dish (plate) can be used; and that no reducing agents toxic to bacteria need be used". The patentee meets this criteria in his own invention by providing a special, thin elastic seal member for fitting between the lid and the base components of a conventional Petri dish. In other words, the patentee modifies the Petri dish with a special seal component.
In the present invention, the desired criteria established by the patentee in U.S. Pat. No. 3,248,302 is also fully met, but in a simpler fashion without the need for a special elastic membrane component to function as a seal, i.e.; without the need to modify a Petri dish.
Sodium hydrosulfite has long been used as an additive or agent in food preservation and is recognized as an antimicrobial; see for example Kirk-Othmer, Encylcopedia of Chemical Technology, Second Edition, Vol. 10, page 13. It has also been used as an ingredient of the well-known Clausen medium.
Clausen medium [dithionite-thioglycollate (HS-T) broth]is a well-known liquid medium used by microbiologists for sterility testing. It includes with a long list of nutritional ingredients in agar, 0.4 gms./liter of sodium hydrosulfite. The medium has a pH of about 7.1 and is sterile by virtue of autoclaving at 121.degree. C. for at least 15 minutes. Autoclaving conditions are known to promote a rapid oxidation or degradation of sodium hydrosulfite and its initial oxidation product sodium metabisulfite.
Under the conditions of use in the present invention, sodium hydrosulfite does not function as an antimicrobial agent and does not appear to generate sulfur dioxide gas,